Light-Induced Printing of Protein Structures on Membranes in Vitro

Abstract

Reconstituting functional modules of biological systems in vitro is an important yet challenging goal of bottom-up synthetic biology, in particular with respect to their precise spatiotemporal regulation. One of the most desirable external control parameters for the engineering of biological systems is visible light, owing to its specificity and ease of defined application in space and time. Here we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on time scales of seconds, and the pattern density can be precisely controlled by protein concentration, laser power, and activation time. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. Thus, light mediated sequential protein assembly in cell-free systems represents a promising approach to hierarchically building up the next level of complexity toward a minimal cell.

One-pot synthesis of prenylated proteins utilizing E. coli cell-free expression

“…Furthermore, targeting to the membrane could be timed by withholding the prenyl donor resulting in a switch-like membrane binding behavior, which can be highly beneficial for the study of signaling cascades. This property offers an added advantage over previously used reconstitution strategies such as including nickelated or biotinylated lipids in the membrane and adding a His tag or Strep tag, respectively, to the protein of interest 61, 62 .…”

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives

“…Blue light triggers ePDZ‐LOVpep binding, which recruits the microtubule to the surface for directional movement. Maximum association between microtubule and motor proteins occurs 6 s after blue light exposure, and dissociation occurs in the dark with a half‐life of 13 s. [ 159 ] Apart from the microtubule dynamics, photoactivatable cell‐free systems can be applied to cell motility, [ 160 ] pattern formation, [ 161 ] and cell interaction. [ 162 ]…”

Fine‐Tuning Protein Self‐Organization by Orthogonal Chemo‐Optogenetic Tools

“…With regard to spatiotemporal control, the use of light to manipulate protein activities is a particularly powerful approach, because the amplitude, wavelength, spatial location, and timing of light illumination can be controlled precisely [6] . Proteins can be spatially targeted and bio‐orthogonally patterned on the membrane by light through genetic fusion, such as light‐inducible chemically modified phospholipid anchors, [7] photoactivatable chemical dimerization, [8] or reversible optogenetic pairs [9] . Moreover, dynamic protein pattern formation can be regulated by photo‐switching the conformation of inhibiting isomeric peptides [10] .…”